Effect of extracellular matrix on bovine spermatogonial stem cells and gene expression of niche factors regulating their development in vitro.

Extracellular matrix (ECM) could influence cells function through providing structural and functional networks facilitating the cellular interactions. The present study was conducted to evaluate the effect of culture on ECM versus plastic on bovine spermatogonial stem cells (SSCs) and growth factors regulating their development. Following isolation, bovine testicular cells were cultured on ECM-coated or uncoated (control) plates for 12 days. The colonization of SSCs was assessed by inverted microscope and the gene expression was evaluated using quantitative real-time PCR. The colonization rate was greater in ECM than the control group (P<0.05). The expression of markers of undifferentiated spermatogonia increased in response to conventional culture (P<0.05). Conversely, the expression of ckit as a marker for differentiated spermatogonia was reduced following culture in the control and ECM groups (P<0.05), but this decrease was less in ECM group (P<0.05). Accordingly, while cells cultured on uncoated plates had greater expression of markers of undifferentiated spermatogonia (P<0.05), cells cultured on ECM-coated plates showed higher expression of ckit (P<0.05). Moreover, culture on ECM resulted in higher expression of kit ligand (Kitlg; P<0.05), whereas culture on plastic led to greater expression of glial cell line-derived neurotrophic factor (Gdnf; P<0.05). In conclusion, the present study revealed that the permissive effect of ECM on bovine SSCs differentiation in vitro, which was probably mediated through upregulation of KITLG expression. Moreover, the results imply that GDNF might contribute to germ cells self-renewal during conventional culture.

Reduced anti-Müllerian hormone (AMH) in mares with hemorrhagic anovulatory follicles.

Anti-Müllerian hormone (AMH) has been observed to decrease with the development of hemorrhagic anovulatory follicles (HAFs) in mares. Two studies were conducted to evaluate AMH concentration in mares with HAFs compared to seasonally anoestrous and cyclic mares, and to elucidate changes of AMH with the development of luteinised unruptured follicles (LUFs). In study 1, AMH and progesterone were evaluated in seasonally anoestrous, anovulatory (with HAF) and cyclic mares (at mid luteal phase). In study 2, mares in control and LUF groups were treated with 1500 IU/case hCG when they had a ≥32-mm follicle and an endometrial oedema score of three (day 0). Mares in the control group received no further treatment. Mares in the LUF group received 1.7 mg/kg flunixin meglumine at the time of hCG administration, and 12, 24 and 36 h after it. Ultrasonography and blood collection for AMH and progesterone measurement were performed on days 0, 1, 2, 4, 6 and 8. In study 1, AMH concentration was lower in seasonally anoestrous and HAF mares than cyclic mares (P<0.05). Progesterone concentration did not differ between HAF and cyclic mares (P>0.05). In study 2, AMH was not different between LUF and control mares (P>0.05); however, progesterone had a lower concentration and a delayed rise after hCG administration in LUF mares compared with the control group (P<0.05). The results indicated that similar to seasonally anoestrous mares, AMH concentrations decreased in mares with HAFs. LUFs were also found to be functionally different from HAFs.

Parallel distribution of sexes within left and right uterine horns in Holstein dairy cows: evidence that the effect of side of pregnancy on sex ratio could be breed-specific in cattle.

Dissimilar distribution of male and female calves within left and right uterine horns has been observed in beef cows. A retrospective study was conducted to investigate the effect of side of pregnancy on secondary sex ratio in Holstein dairy cows. Data associated with sex of calves, side of pregnancy, sire, dam, parity number of dam, AI technician, season and year were retrieved from the database of a Holstein dairy farm. In total, data consisted of 6515 birth records from 3155 dams and 244 sires across years 2001-2010. Data were analyzed using logistic regression. There was no difference in proportion of male and female calves between left (52.9% and 47.1%, respectively) and right (53.2% and 46.8%, respectively) uterine horns (P>0.05). AI technician, year, season and parity of dam did not affect secondary sex ratio (P>0.05). Secondary sex ratio of left and right uterine horns, and consequently, overall secondary sex ratio (53.1%) were skewed toward males as compared with hypothetical secondary sex ratio of 50% (P<0.05). Incidence of right pregnancy (60.5%) was higher than hypothetical 50% incidence of right pregnancy. In conclusion, the present study revealed similar secondary sex ratio of calves between left and right uterine horns in Holstein dairy cows.

More male calves born after Presynch-Ovsynch protocol with 24-hour timed AI in dairy cows.

Various factors including synchronization treatments have been reported to influence sex ratio of offspring in cattle. The present study was conducted to investigate the effect of treatment with Presynch-Ovsynch protocol on sex ratio of offspring in Holstein dairy cows. Healthy Holstein cows (N = 1102) were randomly assigned to Presynch-Ovsynch (N = 564) or control (N = 538) group by parity. Cows in Presynch-Ovsynch group received two administrations of PGF(2)α 14 days apart started at Days 23 to 27 postpartum. Twelve days after the second PGF(2)α treatment, Ovsynch protocol, consisting of an administration of GnRH followed by administration of PGF(2)α 7 days later and a final administration of GnRH given 48 hours after the third PGF(2)α treatment, began. Cows were subjected to fixed-time AI 24 hours after the last administration of GnRH. Cows in control group received no treatment and were inseminated 12 hours after standing estrus. Sex of calves conceived by the first service postpartum was determined after parturition and used for calculation of sex ratio. Parity, season, sire, and calving to conception interval were considered as covariates in statistical analysis. Sex ratio of calves in Presynch-Ovsynch group (1.64) was higher than that in control group (1.09; odds ratio = 1.51; P < 0.05). Moreover, male to female ratio was higher in cows conceived in summer, fall, and winter than in cows that conceived in spring (P < 0.05). In conclusion, cows treated with Presynch-Ovsynch protocol and inseminated 24 hours after the last GnRH administration of Ovsynch had a higher sex ratio than cows inseminated 12 hours after standing estrus.

Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum.

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca(2+) storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca(2+) that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca(2+) store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca(2+) uptake. Direct measurements with ER-targeted Ca(2+)-sensitive fluorescent "cameleon" proteins revealed that Bcl-2 decreased the free Ca(2+) concentration within the lumen of the ER, [Ca(2+)](ER). Analysis of the kinetics of Ca(2+) store depletion in response to the Ca(2+)-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca(2+) concentration within the ER lumen by increasing the Ca(2+) permeability of the ER membrane. The increased ER Ca(2+) permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.

Store-operated Ca2+ influx and stimulation of exocytosis in HL-60 granulocytes.

This study addresses the role of store-operated Ca2+ influx in the regulation of exocytosis in inflammatory cells. In HL-60 granulocytes, which do not possess voltage-operated Ca2+ channels, the chemotactic peptide fMet-Leu-Phe (fMLP) was able to stimulate store-operated Ca2+ influx and to trigger exocytosis of primary granules. An efficient triggering of exocytosis by fMLP required the presence of extracellular Ca2+ and was inhibited by blockers of store-operated Ca2+ influx. However, receptor-independent activation of store-operated Ca2+ influx through thapsigargin did not trigger exocytosis. fMLP was unable to stimulate exocytosis in the absence of cytosolic free Ca2+ concentration [Ca2+]c elevations. However, a second signal generated by fMLP synergized with store-operated Ca2+ influx to trigger exocytosis and led to a left shift of the exocytosis/[Ca2+]c relationship in ionomycin-stimulated cells. The synergistic fMLP-generated signaling cascade was long-lasting, involved a pertussis toxin-sensitive G protein and a phosphatidylinositol 3-kinase. In summary, store-operated Ca2+ influx is crucial for the efficient triggering of exocytosis in HL-60 granulocytes, but, as opposed to Ca2+ influx through voltage-operated Ca2+ channels in neurons, it is not a sufficient stimulus by itself and requires synergistic receptor-generated signals.

Chemoattractant-induced respiratory burst: increases in cytosolic Ca2+ concentrations are essential and synergize with a kinetically distinct second signal.

The role of the cytosolic free Ca2+ concentration ([Ca2+]c) and its relationship to other second messengers in the signalling between chemoattractant [e.g. N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)] receptors and the NADPH oxidase is still poorly understood. In this study, we have used thapsigargin, an inhibitor of the Ca2+-ATPase of intracellular stores, as a tool to selectively manipulate Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane. We thereby temporarily separated the Ca2+ signal from other signals generated by fMLP and analysed the consequences on the respiratory burst. Under all conditions investigated, the extent of fMLP-induced respiratory burst activation was critically determined by [Ca2+]c elevation. fMLP was unable to activate the respiratory burst without [Ca2+]c elevation. Thapsigargin-induced Ca2+ influx activated the respiratory burst in the absence of fMLP, but only to approx. 20% of the values observed in the presence of fMLP. The second signal generated by fMLP did not activate the respiratory burst by itself, but acted in synergy with [Ca2+]c elevation. The second signal was long lasting (>15 min) provided that there was no rise in [Ca2+]c and that the receptor was continuously occupied. The second signal was inactivated by high [Ca2+]c elevation. Our results demonstrate that [Ca2+]c elevations are an essential step in the signalling between the fMLP receptor and NADPH oxidase. They also provide novel information about the properties of the second Ca2+-independent signal that activates the respiratory burst in synergy with [Ca2+]c.